Chapt. 21 oxidative phosphorylation Ch. 21 oxidative phosphorylation Student Learning Outcomes : Explain process of generation of ATP by oxidative phosphorylation: NADH + FAD(2H) donate e- to O 2 -> H 2 O ATP synthase makes ATP (~3/NADH, ~2/FAD(2H) Describe chemiosmotic model, H+ gradient Describe complications of deficiency of ETC –  anemia, cyanide, OXPHOS diseases Describe transport through mitochondrial membranes

I. Oxidative phosphorylation summary Oxidative phosphorylation overview: Multisubunit complexes I, coQ, III, IV pass e- to O 2 H + are pumped out -> electrochemical gradient H + back in through ATP synthase makes ATP Fig. 1

Proton Motive Force Proton motive force : Electrochemical potential gradient Membrane is impermeable to H+ pH gradient ~ 0.75 pH units Fig. 2

ATP synthase Figs. 3,4 ATP synthase (F 0 F 1 ATPase): F 0 inner membrane (12 C) F 1 matrix has stalk, headpiece H+ go through a-c channel 12 protons/turn -> 3 ATP Binding change mechanism : Turning releases ATP

B. Components of Electron Transport Chain Components of Electron Transport chain: Series of transfers of e- down energy gradient Series of oxidation reduction reactions e- finally to O 2 -> H 2 O H+ pushed across membrane Fig. 5

Components of Electron Transport Chain NADH dehydrogenase: 42 subunits, FMN binding proteins Fe-S binding proteins (transfer single e-) binding site for CoQ pass e- to CoQ; transfers 4 H + Fig. 5,6

Components of Electron Transport Chain Complex II: succinate dehydrogenase (from TCA) FAD bound e- from TCA, Other FAD from other paths Not sufficient energy to transfer H+ when pass e- to CoQ Fig. 5

Coenzyme Q Coenzyme Q is not protein bound. 50-C chain inserts in membrane, diffuses in lipid layer Also called ubiquionone (ubiquitous in species) Transfer of single e- makes it site for generation of toxic oxygen free radicals in body Fig. 7

Cytochromes have heme groups Cytochromes have heme groups: Proteins with hemes Fe 3+ -> Fe 2+ as gain e- Transfer e- to lower potential Figs. 5,8; Heme A is in Cyt a, Cyt a 3

C. Pumping of protons not well understood Cytochrome C oxidase: Cyt a, Cyt a 3, O 2 binding: Receives e- from Cyt c (takes 4 to make 2 H 2 O) Transfers to O 2 ; Pumping of H + not well understood; must couple to e- transport and ATP; otherwise backup Fig. 5

D. Energy yield Energy yield from oxidation by O 2 : NADH: Dg0’ ~ -53 kcal; FAD(2H) ~ -41 kcal Each NADH 2e- -> ~ 10 H + pumped; Takes ~ 4 H + /ATP -> 2.5 ATP/ NADH; 1.5/ FAD(2H) or if ~ 3H + /ATP -> 3 ATP/ NADH; 2/ FAD(2H) Fig. 5

E. Inhibition of chain, sequential transfer Once start ETC, must complete transfer of e- In absence of O 2, backup since carriers full of e- Inhibitors like cyanide (binds Cyt c oxidase) mimics anoxia: prevents proton pumping Cyanide binds Fe 3+ in heme of Cyt a a 3 CN in soil, air, foods (almonds, apricots) Fig. 5

OXPHOS diseases from mutated mitochondrial DNA Human mt DNA is kb: 13 subunits of ETC: 7 of 42 of Complex I 1 of 11 Complex III 2 of ATPsynthase 22 tRNA, 2 rRNA OXPHOS diseases from mutated Mitochondrial DNA

Table 21.1 examples OXPHOS diseases from mt DNA Point mutations in tRNA or ribosomal RNA genes: MERFF (myoclonic epilepsy and ragged red fiber): tRNA lys progressive myoclonic epilepsy, mitochondrial myopathy with raged red fibers, slowly progressive dementia Severity of disease correlated with proportion mutant mtDNA LHON (Leber’s hereditary optic neuropathy): 90% of cases from mutation in NADH dehydrogenase Late onset, acute optic atrophy

Nuclear genes can cause OXPHOS Mutated nuclear genes can cause OXPHOS: About 1000 proteins needed for Oxidation phosphorylation are encoded by nuclear DNA. Electron transport chain, translocators Need coordinate regulation of expression of genes, import of proteins into mitochondria, regulation of mitochondrial fission Nuclear regulatory factors for transcription in nucleus, mt Often recessive autosomal

III. Coupling of electron transport and ATP synthesis Fig. 10 Concentration of ADP controls O2 consumption: Or phosphate potential ( [ATP]/[ADP][Pi]) 1.ADP used to form ATP 2.Release ATP requires H + flow 3.H + decreases proton gradient 4.ETC pumps more H +, uses O 2 5.NADH donates e-, makes NAD+ to return to TCA cycle or other

Uncoupling agents dissipate H+ gradient without ATP Uncoupling agents decrease H+ gradient without generating ATP: Ex. DNP is a chemical uncoupler: lipid soluble, carries H + across membrane Fig. 11

Uncoupling proteins form channels, thermogenesis Uncoupling proteins form channels for protons: Ex. UCP1 (thermogenin) makes heat in brown adipose tissue (nonshivering thermogenesis); many mitochondria; Infants have lots of brown adipose tissue, not adults Fig. 12

IV. Transport through mitochondrial membranes Transport across inner mitochondrial membranes uses channels, translocases: Form of active transport using proton gradient : ANT exchanges ATP: ADP Symport H + with Pi Symport H +, pyruvate Fig. 13

Transport across outer membrane: Fig. 13 Transport across outer membrane: Rather nonspecific pores: VDAC voltage-dependent anion channels Often kinases on cytosolic side

Mitochondrial permeability transition pore Fig. 14 Mitochondrial permeability transition pore: Large nonspecific pore: Will lead to apoptosis (cell death) Highly regulated process Hypoxia can trigger Pore opens, lets H+ flood in, Anions, cations enter Mitochondria swell and Irreversible damage

Key concepts Reduced cofactors NADH, FAD(2H) donate e- to electron transport chain ETC transfers e- to O2 -> H2O As e- transferred, H+ pushed across membrane; H+ gradient used by ATP synthase to make ATP O2 consumption tightly coupled to ATP synthesis Uncouplers disrupt process – poisons OXPHOS diseases from mutations in mt DNA or in nuclear DNA Compounds transported across mt membranes

Review question 5. Which of the following would be expected for a patient with an OXPHOS disease? A.A high ATP:ADP ratio in the mitochondria B.A high NADH:NAD+ ratio in the mitochondria C.A deletion on the X chromosome D.A high activity of complex II of the electron-transport chain E. A defect in the integrity of the inner mitochondrial membrane

Review question p. 392 Decreased activity of the electron transport chain can result from inhibitors as well as from mutations in DAN. Why does impairment of the ETC result in lactic acidosis? Inhibit ETC -> Impaired oxidation of pyruvate, fatty acids and other fuels; therefore more lactate and pyruvate in blood. NADH oxidation requires complete transfer of e- to O2, so defect in chain increase NADH:NAD+ and inhibit pyruvate dehydrogenase.